阅读说明：本技术 一种苦味抑制肽及其应用 (Bitter taste inhibitory peptide and application thereof ) 是由 励建荣 李冬慧 赵文竹 于志鹏 于 2020-11-18 设计创作，主要内容包括：本发明保护了一种苦味抑制肽Gln-Arg-Pro-Arg在制备具有掩盖苦味作用的苦味抑制剂中的应用。本发明所提供的具有苦味抑制作用的生物活性肽Gln-Arg-Pro-Arg可与人类苦味受体TAS2R14活性位点结合,对苦味有很好的抑制效果,且具有安全无毒副作用,水溶性良好等特点,解决了现有技术苦味抑制效果不明显等技术问题,可作为苦味抑制剂用于食品、保健品以及药品中,具有广泛的应用前景和十分重要的意义。(The invention protects the application of a bitter taste inhibitory peptide Gln-Arg-Pro-Arg in preparing a bitter taste inhibitor with the bitter taste masking effect. The bioactive peptide Gln-Arg-Pro-Arg with the bitter suppression effect can be combined with the active site of a human bitter receptor TAS2R14, has a good bitter suppression effect, has the characteristics of safety, no toxic or side effect, good water solubility and the like, solves the technical problems of unobvious bitter suppression effect and the like in the prior art, can be used as a bitter inhibitor for foods, health-care products and medicines, and has wide application prospect and very important significance.)

1. Application of bitter taste inhibiting peptide Gln-Arg-Pro-Arg in preparation of bitter taste inhibitor with bitter taste masking effect is provided.

Technical Field

The invention belongs to the field of bioactive peptides, and particularly relates to a bitter taste inhibitory peptide and application thereof.

Background

Taste is a very important perception of mammals, bitterness is one of five basic tastes, the threshold is very low and lasts longer than other tastes, and many natural organic substances including caffeine, nicotine, brucine and numerous chemically synthesized drugs are bitter substances. A relatively weak bitter taste may cause unpleasant sensations, while a strong bitter taste may cause nausea, vomiting and psychological aversion, and many consumers may refuse to purchase the food product because of their own bitter taste. It has been found that many functional ingredients in foods, health products and pharmaceuticals have a bitter taste, for example, a bitter component hop extract contained in beer, which has sedative and stomachic effects; catechins and tannins contained in tea and wine; caffeine and chlorogenic acid contained in coffee in a large amount; naringin and hesperidin contained in citrus fruit; proteins such as casein and whey and peptides, amino acids as degradation products thereof; various vitamins such as vitamin B1; trace minerals such as calcium, magnesium, etc.; quinine, berberine and the like in the alkaline medicines. Although a slight bitterness can effectively improve palatability and food flavor, if the bitterness is too strong, unpleasant feeling or aversion feeling may be accompanied. Therefore, research on methods for eliminating or suppressing bitter taste is also an important point in the development of foods, beverages, health products, and pharmaceuticals.

Bitter taste is sensed by the fact that bitter substances dissolved in saliva act on receptors on microvilli of taste cells, depolarize cell membranes and release neurotransmitters through intracellular signal transduction, and then excitatory signals are transmitted to subcortical centers through postsynaptic afferent nerve fibers and are integrated through the subcortical centers. Bitter taste is mediated by the bitter taste receptor TAS2Rs, which belongs to the superfamily of G protein-coupled receptors. TAS2R14(T2R14) is one of the most widely tuned bitter receptors for a variety of bitter compounds, and it recognizes a wide variety of natural or synthetic bitter compounds, including many drugs. Therefore, T2R14 can be used as an effective target for inhibiting bitter taste.

Bitterness suppression refers to reducing or eliminating the bitterness of bitter substances. At present, various methods for suppressing bitter taste have been reported at home and abroad, and the most common method is to add an inhibitor or a retarder to mask or eliminate the bitter taste. For example, common salt has an inhibitory effect on many bitter substances (quinine, caffeine, urea, etc.); various flavonoids such as sakuranetin, 6-methoxy sakuranetin, and jaceosidin can inhibit bitter taste receptor TAS2R10 activity; some carboxylic acid compounds can obviously inhibit the activation of the bitter taste receptor TAS2R16 by salicin, but the action conditions of the inhibitors are difficult to control and can be possibly influenced with functional components in food, health care products or medicines. The selective separation method is mainly used for removing bitter peptides, and the bitter peptides are removed by adopting the adsorption effect of activated carbon, glucan, agar, glass fiber and the like, or extracted by using some organic solvents such as ethanol, propanol and the like, and the process can influence the flavor of food and the absorption of polypeptide by human body. In addition, the adoption of ultrafiltration technology treatment is also an effective method for debitterizing, bitter peptides can be removed by using several ultrafiltration membranes with different molecular weight cut-off ranges, but the application range is smaller, and the method is only applied to debitterizing soybeans at present. The enzyme method adopts exo-polypeptidase to remove hydrophobic amino acids at two ends of bitter peptide to achieve the purpose of debittering, but the method has higher cost and less application.

The food-derived bioactive peptide has good bioactivity and functionality, high safety, no toxic or side effect and easy digestion and absorption, and the research of inhibiting bitter taste by utilizing the competitive principle of the food-derived bioactive peptide and the taste receptor draws attention of a large number of scholars. The food-derived active peptide can provide human body nutrition requirements, and can also solve the influence caused by undesirable bitter taste in foods, medicines and health products.

The invention aims to provide a bitter taste inhibitory peptide and application thereof in covering undesirable bitter taste in foods, medicaments and health-care products.

Disclosure of Invention

The present invention has been made to overcome the above-described drawbacks of the prior art, and an object of the present invention is to provide a bitter taste-suppressing peptide and a method for suppressing bitter taste.

The bitter taste inhibitory peptide has an amino acid sequence of Gln-Arg-Pro-Arg, has a powdery crystal, and has a half Inhibitory Concentration (IC) for inhibiting the activity of bitter taste receptor T2R14₅₀) 270 μ M.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the present invention provides a bitter taste inhibitory peptide derived from patinopecten yessoensis myosin.

In another aspect, the present invention provides a method for obtaining bitter taste inhibitory peptides, using an online database for multiple rounds of screening, comprising the steps of:

(1) and searching a related protein sequence of the patinopecten yessoensis from an NCBI database for analysis, and selecting the availability of sequence information according to the patinopecten yessoensis myosin.

(2) Virtual hydrolysis of myosin by ExPASY PeptideCutter (http:// web. ExPASy. org/peptide _ cutter /) using pepsin, chymotrypsin and trypsin, selection of all polypeptides, selection of partial polypeptides with scores above 0.5 in the online program PeptideRanker (http:// bioarea. ie/. about.sub./compass/bioarea web/Server _ pages/Pept idenderker. php), prediction of water solubility using the online tool Innovagen (http:// www.innovagen.com/proteomics-tools), ADMET property prediction using Discovery Studio 2017, final selection of polypeptides with good water solubility, no toxicity and high biological activity, docking with the bitter receptor T2R14 in DS2017, selection of docking energy values for lower peptide sequences, in vitro validation of bitter inhibitory activity.

(3) Molecular docking was used to predict T2R14 interaction with the peptide. In this experiment, the crystal structure of T2R14 was obtained from the BitterDB database. Molecular docking was performed using DS2017 software and used to analyze peptide interactions at the binding sites. The final analysis was done manually. Prior to the analysis, the following steps were performed: (i) using a CDOCKER docking program; (ii) selecting a CHARMM force field; (iii) the active center of T2R14 is XYZ (1.19548, 2.03435, 11.9968, 17.4). CHARMm force field was used to optimize the conformation of each peptide. And screening to obtain the potential active tetrapeptide QRPR with bitterness inhibiting effect by taking the 'CDOCKER-INTERACTION-ENERGY' score shown by the docking result as an index.

(4) In vitro bitterness inhibition activity of active peptides QRPs. 30mL of active peptide QRPR solutions with different concentrations are prepared, 50mL of bitter substance solution with certain concentration is added to prepare solution to be detected, the bitter substance solution is used as a blank control group, and the peptide LEGSLE is used as a positive control group. Calculating bitter taste inhibition ratio of active peptide with different concentrations, and obtaining IC of active peptide for inhibiting bitter taste receptor₅₀The value is obtained. The bitter substance may be quinine or mandarin orangeBitter components such as fructus Citri Tangerinae, tea, cocoa and flos Lupuli, or Chinese medicinal materials such as Coptidis rhizoma, cortex Phellodendri, rhizoma Atractylodis and radix Gentianae.

The invention has the following beneficial effects: the invention provides a bitter suppression peptide, which is obtained by screening a peptide QRPR capable of effectively suppressing bitter from patinopecten yessoensis myosin and defines action sites of the peptide QRPR and a bitter receptor T2R 14; the QRPR has good bitterness inhibiting effect, good bitterness inhibiting effect on bitter substances, safety, no toxicity, good water solubility and biological activity, and compared with the bitter inhibitor with the currently disclosed compound proportion, the QRPR has simple application and operation, does not depend on complex technology, can obviously improve the taste of food and improve the flavor, and can be used as the bitter inhibitor to be applied to food, health care products and medicines, thereby having good potential and application prospect.

Drawings

The invention is illustrated in figure 1, wherein:

fig. 1 is a schematic 3D spatial interaction of QRPR with bitter receptor T2R 14;

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1 screening of bioactive peptides having bitter taste inhibitory Effect

1. Screening of bitter taste inhibitory peptides

The method carries out virtual enzymolysis on the myosin sequence of the Japanese scallop by using an online enzymolysis tool of ExPASY PeptideHouter (http:// web. ExPASy. org/peptide _ cutter), and 323 polypeptides are obtained after enzymolysis. 323 polypeptides are screened for bioactivity and water solubility in the threading order by peptide property calulator and PeptideRanker. Toxicity prediction using ADMET program of DS2017 software, three toxicity properties were predicted: mutagenicity, developmental toxicity and skin sensitization. 10 safe and nontoxic polypeptides with good water solubility and bioactivity score higher than 0.5 are obtained, and the sequences are SM, RR, ADM, GFPSR, GIR, KPM, QRPR, NPPK, DM and DPDF respectively.

2. Prediction and analysis of bitter taste suppression Activity

The crystal structure of the bitter receptor T2R14 was obtained from the BitterDB database (http:// BitterDB. agri. huji. ac. il/dbbitter. php), the small molecule ligand was removed, the hydrogen atom and CHARMM were added in the vertical field, and the definition of the active center was performed according to the existing data as the receptor protein. Introducing the screened 10 polypeptides into Discovery Studio 2017, constructing a three-dimensional structure, and optimizing the three-dimensional structure by using Minimization Ligand as a micromolecular Ligand. The treated receptor protein and the small molecule ligand are subjected to molecular docking through a CDOCKER program in DS2017 to screen a polypeptide capable of tightly binding with T2R14, and an active site is defined. And screening the active tetrapeptide QRPR with good bitterness inhibition effect by taking the 'CDOCKER-INTERACTION-ENERGY' score shown by the docking result as an index. The DS software is used for analyzing the INTERACTION mode between the active peptide QRPR and the bitter taste receptor T2R14, and the 'CDOCKER-INTERACTION-ENERGY' ENERGY value of the bitter taste inhibitory peptide QRPR is 77.2643. The 3D spatial interaction of QRPR with receptor binding is shown in fig. 1, and it can be seen from fig. 1 that: best docking posture of QRPR with bitter receptor T2R 14; QRPR interacts with Typ159, Asn157, Gly158, Trp89, Phe175, Ser167 residues of the bitter receptor T2R14, binding to Typ159, Trp89 and Phe75 through hydrophobic interactions, and binding to Asn157, Gly158 and Ser67 through traditional hydrogen bonding.

Example 2 demonstration of bitter inhibitory peptide QRPs for bitter inhibitory Activity

The peptide QRPR is prepared by means of solid phase synthesis, fermentation or enzymolysis, freeze-dried powder of the peptide is dissolved in deionized water to prepare solutions with different solubilities, 50mL of 1mM quinine solution is added to prepare a solution to be detected, reported bitterness inhibiting peptide LEGSLE is used as a positive control, the solutions are respectively filled into a special beaker for an electronic tongue and placed in an automatic sample introduction analysis device, and the change of membrane potential is detected. The result shows that the inhibition rate of 0.15mg/mL active peptide QRPR on the bitterness of quinine is 50%, and the inhibition effect is stronger than that of a positive control.

Meanwhile, the bitterness-inhibiting peptide QRPR can also be used as a bitterness inhibitor for foods containing bitter components such as citrus, tea, cocoa and hop, or for Chinese medicinal materials and health products containing Coptidis rhizoma, cortex Phellodendri, rhizoma Atractylodis and radix Gentianae to inhibit bitterness.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.